Lysosomes, endosomes, and a variety of other intracellular organelles are acidified by a family of unique proton pumps, termed the vacuolar H(+)-ATPases, that are evolutionarily related to bacterial membrane proton pumps and the F1-F0 H(+)-ATPases that catalyze ATP synthesis in mitochondria and chloroplasts. The electrogenic vacuolar H(+)-ATPase is responsible for generating electrical and chemical gradients across organelle membranes with the magnitude of these gradients ultimately determined by both proton pump regulatory mechanisms and, more importantly, associated ion and organic solute transporters located in vesicle membranes. Analogous to Na+, K(+)-ATPase on the cell membrane, the vacuolar proton pump not only acidifies the vesicle interior but provides a potential energy source for driving a variety of coupled transporters, many of them unique to specific organelles. Although the basic mechanism for organelle acidification is now well understood, it is already apparent that there are many differences in both the function of the proton pump and the associated transporters in different organelles and different cell types. These differences and their physiologic and pathophysiologic implications are exciting areas for future investigation.